Membrane proteins, either alone or in complexes, carry out many important physiological functions in all cells in response to signaling and regulation. The synthesis and assembly of membrane proteins into the hydrophobic lipids phase to carry out various functions have long been intensively investigated. Many functional membrane proteins possess channel activities that could be monitored by electrophysiological methods in response to intracellular or extracellular stimuli or regulatory molecules. Oocyte injection has been widely used for monitoring membrane ion channel activity for years, and is well-known for studying channels in a controlled in vivo cellular environment. By injecting specific mRNA or cDNA into the oocytes, the target channels can be expressed and assembled in the oocyte membranes for whole cell voltage clamp recording (1, 2), patch clamp and other biochemical approaches (3).
This method is exquisitely sensitive because the channels are expressed and presented on the whole oocyte membrane surface; with the channel activity recording being based on the sum of the channel activities in the whole cell, the resulting ion current is detectable at μA levels. Additional advantages of this assay include low endogenous channel activities, large size of the oocytes, and the detection efficiency of target protein activity (4). So far it has been widely employed for analyzing characteristics and regulation of channels, domain mutations, and drug and drug resistance screening (5).
However, there are several major challenges for further application to the study of membrane proteins. First, not all target protein channels can be expressed in the oocytes, and in some cases, the mRNA or cDNA are not available, for example, in the use of clinical samples (6). Second, there is no methodology readily available for the study of complex systems with multiple, membrane protein complexes. Third, most membrane proteins, especially eukaryotic proteins, require special environments for folding, assembly, post-modification, and trafficking. The direct correlation of in vitro liposomes studies to the cellular physiological activities of these membrane proteins has not been achieved despite of the fact that tremendous effort and progress have been made for in vitro refolding and assembly of purified channel membrane proteins, using various liposome technologies. To date, it is not known whether such systems can indeed function for channel activity within an in vivo physiological cellular system. In addition, there is increasing evidence that different compositions of lipids actually modulate channel activities (7). Therefore, there remains a need for additional methods of analyzing characteristics and regulation of membrane proteins, such as channels, pumps, and receptors.
Accordingly, it is an object of the invention to provide compositions and methods for analyzing characteristics and regulation of membrane proteins, such as channels, pumps, and receptors in a physiologically relevant in vitro system.
It is also an object of the invention to provide compositions and methods for screening new compounds that bind to or act on membrane proteins.